Fig 2: Loss of p54nrb leads to an altered expressional pattern of carcinogenesis relevant genes.A Volcano plot diagram of LC/MS data derived from the analysis of HeLa shRNA-control versus shRNA-p54nrb cells. (n = 3). Arrowheads depict significantly (p < 0.01) upregulated (arrow up, red) and downregulated (arrow down, blue) proteins, and the total numbers of both categories are indicated on the diagram. B Up- and C downregulated proteins (p < 0.01, Log2 ratio either >0.5 or < −0.5) of HeLa-shRNA-p54nrb cells compared to shRNA control cells, which exert tumor regulatory and/or apoptosis regulatory properties. Differently colored oval diagrams represent various functional subcategories of these hits. D Immunoblot of HeLa shRNA-control and shRNA-p54nrb cells. Detection of p54nrb, gelsolin, cathepsin-Z, NQO1, and TPD52 levels. E Quantitative evaluation of immunoblots of p54nrb, gelsolin, cathepsin-Z, NQO1, TPD52, and CDKN2A from HeLa shRNA-control and shRNA-p54nrb#1 and #3 cells. The bands were normalized to the shRNA-control samples. Significance was calculated with one sample t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 3.

Fig 3: NONO preferentially binds GGGGCC repeat RNA in the G-quadruplex conformation. (A) Domain architecture of DBHS proteins and NONO(53-312) construct with shared DBHS core region colored by domain and low-complexity regions in grey. (B) EMSA with recombinant NONO(53-312) and (GGGGCC)4 RNA in G-quadruplex and non-G-quadruplex conformations. 10 nM labelled (GGGGCC)4 RNA was incubated with NONO(53-312) at the following concentrations (left to right): 0, 1.2 nM, 6.0 nM, 12 nM, 24 nM, 30 nM, 60 nM, 120 nM, 150 nM, 200 nM, 300 nM, 600 nM, 750 nM, 1.0 µM, 1.5 µM, 3.0 µM, 5.0 µM. For visualization purposes, images were adjusted independently to account for differences in fluorescence intensity between conformations. Quantification was performed prior to this adjustment. (C) Data fit of (B) with binding curves and SEM (n = 3). In this and subsequent figures, error bars for some data points are within the size of the symbol and are therefore not visible.

Fig 4: GGGGCC repeats recruit NONO to model transcripts. (A) An eGFP-encoding model transcript system was used to test whether the presence of GGGGCC repeat sequence affects NONO-transcript association in a cellular context. Constructs contained an eGFP coding sequence transcribed under the control of a cytomegalovirus (CMV) promoter and bovine growth hormone polyadenylation signal (PAS), with experimental sequences inserted in the 5'-untranslated region. Two control transcripts were used: the Spacer construct included a 25-nucleotide spacer insert and the (TGGTCC)8 construct included eight TGGTCC repeats, providing a mutated repeat sequence incapable of forming G-quadruplex structures. (B) RNA-IP for NONO was performed on HEK293T cells transfected with the model transcript constructs, followed by RT-qPCR to compare enrichment of the model transcripts. Transcript enrichment is expressed as percentage of input with SEM (n = 3). ‘*’ indicates P < 0.05 (two-tailed t-test). (C) Western blot of NONO IP fractions, 5% input, and enriched fraction from IP with IgG control. Actin is blotted as a negative control for IP enrichment.

Fig 5: MM patients exhibit high levels of RNA: DNA hybrids and IFN activation (related to Fig 7) Data from Agnelli et al (2009) provided by Oncomine database and reanalyzed for the expression of NEAT1, SFPQ and NONO, in normal bone marrow, MGUS, MM, and plasma cell leukemia (n = 158). The associate P‐value is shown above Box and whisker plot shows the upper and lower quartiles (25–75%) with a line at the median, whiskers extend from 10th to the 90th percentile, and dots correspond to the minimal and maximal values.Boxplot showing the relative enrichment of paraspeckles genes (Right) and NEAT1 (Left), derived from the transcriptomes of the selected CoMMpass patient cohort (N = 687), by the relative ISS score. The enrichment values reported on y‐axis correspond to raw reads counts normalized with weighted trimmed mean of M‐values (TMM). The center line denotes the median value (50th percentile) while the boxes contain the 25th to 75th percentiles of data. The whiskers mark the 5th and 95th percentiles.Kaplan–Meier survival curves of overall survival (OS) in the CoMMpass cohort (N = 687) with IFN signature selection expression on a continuous scale. The patient cohort was divided in IFN high (N = 341) (red) and IFN low (N = 291) (black) by splitting the population at the IFN signature median threshold. The patients above the IFN median were assigned to IFN High, while patients below the median were assigned to IFN low group.Kaplan–Meier survival curves of overall survival (OS) in the CoMMpass cohort (N = 687) with IFN signature selection on a discrete scale. The patient cohort was split in IFN highest (N = 71) (red) and the remaining MM cohort (N = 615) (black) as described in the methods section.